1. Field of the Invention
The present invention relates to a chondroitin synthase gene and methods of making and using same. In more particular, but not by way of limitation, the present invention relates to a chondroitin synthase gene from Pasteurella multocida and methods of using same in a gram-positive host.
2. Background Information Relating to the Invention
Glycosaminoglycans [GAGs] are long linear polysaccharides consisting of disaccharide repeats that contain an amino sugar and are found in most animals. Chondroitin [β(1, 4)GlcUA-β(1,3)GalNAc]n, heparin/heparan [α(1,4)GlcUA-β(1, 4)GlcNAc]n, and hyaluronan [β(1, 4)GlcUA-β(1, 3)GlcNAc]n are the three most prevalent GAGs found in humans. Chondroitin and heparin in animals typically have n=20 to 100, while hyaluronan typically has n=103. Chondroitin and heparin are synthesized as glycoproteins and are sulfated at various positions in vertebrates. Hyaluronan is not sulfated in vertebrates. A substantial fraction of the GlcUA residues of heparin and chondroitin are epimerized to form iduronic acid.
Many lower animals possess these same GAGs or very similar molecules. GAGs play both structural and recognition roles on the cell surface and in the extracellular matrix. By virtue of their physical characteristics, namely a high negative charge density and a multitude of polar hydroxyl groups, GAGs help hydrate and expand tissues. Numerous proteins bind selectively to one or more of the GAGs. Thus the proteins found on cell surfaces or the associated extracellular matrices (e.g., CD44, collagen, fibronectin) of different cell types may adhere or interact via a GAG intermediate. Also GAGs may sequester or bind certain proteins (e.g. growth or coagulation factors) to cell surfaces.
Certain pathogenic bacteria produce an extracellular polysaccharide coating, called a capsule, which serves as a virulence factor. In a few cases, the capsule is composed of GAG or GAG-like polymers. As the microbial polysaccharide is identical or very similar to the host GAG, the antibody response is either very limited or non-existent. The capsule is thought to assist in the evasion of host defenses such as phagocytosis and complement. Examples of this clever strategy of molecular camouflage are the production of an authentic HA polysaccharide by Gram-negative Type A Pasteurella multocida and Gram-positive Group A and C Streptococcus. The HA capsule of these microbes increases virulence by 102 to 103-fold as measured by LD50 values, the number of colony forming units that will kill 50% of the test animals after bacterial challenge.
The invasiveness and pathogenicity of certain E. coli strains has also been attributed to their polysaccharide capsules. Two Escherichia coli capsular types, K4 and K5, make polymers composed of GAG-like polymers. The E. coli K4 polymer is an unsulfated chondroitin backbone decorated with fructose side-branches on the C3 position of the GIcUA residues. The E. coli K5 capsular material is a polysaccharide, called heparosan, identical to mammalian heparin except that the bacterial polymer is unsulfated and there is no epimerization of GlcUA to iduronic acid.
The studies of GAG biosynthesis have been instrumental in understanding polysaccharide production in general. The HA synthases were the first GAG glycosyltransferases to be identified at the molecular level. These enzymes utilize UDP-sugar nucleotide substrates to produce large polymers containing thousands of disaccharide repeats. The genes encoding bacterial, vertebrate, and viral HAS enzymes have been cloned. In all these cases, expression studies demonstrated that transformation with DNA encoding a single HAS polypeptide conferred the ability of foreign hosts to synthesize HA. Except for the most recent HAS to be identified, P. multocida PmHAS, these proteins have similar amino acid sequences and predicted topology in the membrane. Two classes of HASs have been proposed to exist based on these structural differences as well as potential differences in reaction mechanism
The biochemical study of chondroitin biosynthesis in vertebrates was initiated in the 1960s. Only recently have the mammalian enzymes for elongation of the polysaccharide backbone of chondroitin been tentatively identified by biochemical means. An 80-kDa GlcUA transferase found in vertebrate cartilage and liver was implicated in the biosynthesis of the chondroitin backbone by photoaffinity labeling with an azidoUDP-GlcUA probe. A preparation from bovine serum with the appropriate GalNAc- and GlcUA-transferase activities in vitro was obtained by conventional chromatography, but several bands on SDS polyacrylamide gels (including a few migrating ˜80 kDa) were observed. Several genes called ChSy have been recently identified that encode the enzymes that polymerize the chondroitin backbone in animals and humans; these proteins are not homologous to the Pasteurella PmCS gene described herein.
Chondroitin polysaccharide ([β(1,3)GalNAc-β(1,4)GlcUA]n; where n=˜10-2000) has use as a hyaluronan (HA) polysaccharide substitute in medical or cosmetic applications. Both chondroitin and hyaluronan form viscoelastic gels (suitable for eye or joint applications) or hydrophilic, hygroscopic materials (suitable for moisturizer or wound dressings). Unmodified or underivatized chondroitin is not known to exist or, if present, in very small quantities in the human body. The main advantage is that byproducts of natural HA degradation (by shear, enzyme, radical or oxidation processes) have certain biological activities with respect to vascularization, angiogenesis, cancer, tissue modulation, but similar byproducts of chondroitin (in the proposed unsulfated, unmodified state) may not have the same biological activity. The chondroitin polymers are more inert, loosely speaking, than the analogous HA molecule. Chondroitin from either P. multocida Type F or a recombinant host containing the Pasteurella-derived or Pasteurella-like synthase gene will serve as an alternative biomaterial with unique properties.
With respect to related microbial GAG synthases other than the HASs, the E. coli K5 glycosyltransferases that synthesize heparosan have been identified by genetic and biochemical means. In contrast to the HASs, it appears that two proteins, KfiA and KfiC, are required to transfer the sugars of the disaccharide repeat to the growing polymer chain. The chondroitin-backbone synthesizing enzyme of E. coli K4 has been enzymatically characterized, and the gene encoding the relevant glycosyltransferases, KfoC, was recently discovered; it is very homologous (e.g., DNA will cross-hybridize) to the PmCS gene described herein. The KfoC enzyme performs the same reaction as PmCS in vitro, but the former protein appears less robust.
Many P. multocida isolates produce GAG or GAG-like molecules as assessed by enzymatic degradation and removal of the capsule of living bacterial cells. Type A P. multocida, the major fowl cholera pathogen, makes a capsule that is sensitive to hyaluronidase. Subsequent NMR structural studies of capsular extracts confirmed that HA was the major polysaccharide present. A specific HA-binding protein, aggrecan, also interacts with HA from Type A P. multocida. Two other distinct P. multocida types, a swine pathogen, Type D, and a minor fowl cholera pathogen, Type F, produce polymers that are chondroitin or chondroitin-like based on the observation that their capsules are degraded by Flavobacterium chondroitin AC lyase. After enzymatic removal of the capsule, both types were more readily phagocytosed by neutrophils in vitro. The capsule of Type D cells, but not Type F cells, is also reported to be degraded by heparinase III, suggesting a heparin-type molecule is present, too.
Parent application U.S. Ser. No. 09/842,484 discloses the identification of PmCS (P. multocida Chondroitin Synthase), the first chondroitin synthase to be identified and molecularly cloned from any source. Interestingly, a single polypeptide is responsible for the copolymerization of the GlcUA and GalNAc sugars, and thus PmCS is a single protein that is a dual-action transferase that catalyzes the polymerization of UDP-GlcUA and UDP-GalNAc to form chondroitin. The '484 parent application also identified the Type F capsular polymer as an unsulfated chondroitin polymer, and identified organisms with the chondroitin synthase gene (Type F P. multocida) as new sources of unsulfated chondroitin polymer.
Certain glycosaminoglycan synthase enzymes (namely enzymes from Streptococcus, virus and vertebrates) appear to be self-contained, meaning that they possess both (a) sugar addition or polymerization activity (glycosyltransferase) as well as (b) sugar export functions (transport the polymer across the membrane to the outside of the cell where it can be easily harvested). Certain other distinct enzymes, including those from Pasteurella, can catalyze glycosaminoglycan polymerization, but in general are thought to not be able to complete the sugar export step without assistance of other proteins. Gram-negative bacteria have two membranes and are generally thought to need more transport machinery than Gram-positive bacteria with a single membrane. In addition, Pasteurella (as well as other sugar-producing Gram-negative pathogens such as E. coli) is not a preferred host for the production of glycosaminoglycans intended for use in animals or humans due to the presence of endotoxins or lipopolysaccharides (molecules derived from their outer membrane due to the possibility of inducing shock, etc.
The Gram-negative bacteria capable of GAG biosynthesis, Escherichia coli and Pasteurella multocida, possess two lipid membranes, and their capsule loci encode many transport-associated proteins in addition to the glycosyltransferases and the UDP-GlcUA forming enzymes (˜10-18 kilobases; Roberts, 1996; Townsend et al, 2001). Although many details are not well understood, in the best-studied model, the E. coli Group II capsular system, it appears that transport of the nascent polymer chain requires an apparatus composed of at least 7 distinct polypeptide species (Whitfield and Roberts, 1999; Silver et al, 2001). Briefly, a complex containing KpsC,M,S,T assembles on the inner membrane and interacts with the KfiA,B,C catalytic complex. KpsM and T form the ATP-binding cassette (ABC) transporter. A periplasmic protein, KpsD, and a dimer of another inner membrane protein, KpsE, help transport the polymer across the periplasmic space (Arrecubieta, 2001). A porin complex in the outer membrane is recruited to transport the growing polysaccharide chain out of the cell. Certain Kps mutants polymerize the capsular polysaccharide chain, but possess faulty translocation resulting in polymer accumulation in the cytoplasm or periplasm. P. multocida is also thought to have a Group II-like transport system based on the sequence similarities and gene arrangement of its putative transport proteins to the E. coli proteins.
In the case of PmHAS and PmCS, the carboxyl-terminal tail was thought to contain a docking segment that interacts with the transport mechanism (Jing and DeAngelis, 2000). However, the region(s) of E. coli K5 enzymes responsible for docking to the transport apparatus is not known and there is no obviously similar sequence to the carboxyl-terminus of the Pasteurella enzymes. Polymer transport across membranes is a difficult phenomenon to study. Therefore, it is thought that any recombinant microbial system endeavoring to utilize the Pasteurella glycosaminoglycan synthases must solve or circumvent the transport problem.
Therefore, there is a need felt in the art to provide methods of expressing Gram-negative glycosaminoglycan synthase gene in a Gram-positive bacterial host background to produce GAGs by fermentation in vivo. It is to such methods of expressing a Gram-negative glycosaminoglycan synthase gene in a Gram-positive host that the present invention is directed.